We recently showed that the product of the Drosophila spire gene, p150-Spir (Spir) nucleates actin filament formation by a unique mechanism (Quinlan et al., 2005). We recently discovered that Spir binds directly to members of another class of actin nucleators, the Cappuccino-family formins (Preliminary Results). This result is consistent with previous genetic studies indicating that the two proteins operate in the same pathway to specify both anterior-posterior and dorsal-ventral axes of developing embryos (Manseau and Schupach, 1989) and localization studies demonstrating that they have identical expression patterns in embryonic and adult mammalian tissues (Schumacher et al., 2004). Spir- and Cappuccino-family proteins are conserved across metazoan species from fruitflies to humans and in mammals and every organism known to express a Spir-family protein also expresses a Cappuccino-family formin. Despite their conservation and their importance in development, however, the function and regulation of Spir and Capu are not well understood. Our results suggest that the activities of Spir and Cappuccino are directly coupled and that they may function together in vivo as a single filament-forming complex. Our long-term goal is to understand how these actin nucleation factors work together to determine polarity of oocytes and embryos. Our short-term goals are to understand the activities of Spir and Capu alone and in complex. We will also investigate a potential mechanism by which the two proteins may be targeted in vivo. Specifically, we will: 1. Determine the mechanism of actin nucleation by Spir. 2. Characterize the interaction of Cappuccino with actin. 3. Characterize the interaction between Spir and Cappuccino. 4. Characterize the interaction of the Spir FYVE domain with lipids. [unreadable] [unreadable] [unreadable]